The future for molecular diagnostics

Will whole laboratories one day be contained on a single electronic chip, with all diagnostic testing performed exclusively at the point of care? Such concepts may still seem impossibly futuristic, but when one stops to consider the pace at which laboratory technology is currently evolving, it would surely be short-sighted to dismiss them out of hand

'Current and future trends in Molecular Diagnostics' was the theme of a presentation made by Dr Panagiotis Pantelidis of the Pan Pathology Molecular Diagnostics facility at Guy's and St Thomas' NHS Foundation Trust to delegates at a meeting organised by Bayer Diagnostics (a division of Bayer HealthCare). Those attending Bayer’s annual Molecular User Group Meeting at The Royal Opera House, Covent Garden, gained an insight into Dr Pantelidis’ vision for the future, inspired by today’s rapid development and acceptance of molecular tests.
“Molecular Biology methods are now routinely used in every pathology discipline, and are also now included in National Diagnostic Recommendations”, he said. “This explosion has been driven by the need for faster, accurate diagnostic tests, fuelled by the inception of the Polymerase Chain Reaction (PCR) in 1985. Initially, molecular diagnostic assays were developed due to the close interaction between diagnostic laboratories and academic research centres, but by far the most important factor has been the availability of increased genetic data information for human and pathogenic genomes.”
Dr Pantelidis explained that Virology Departments have been in the forefront of embracing molecular technology because alternative techniques (such as culture) are often lengthy, require constant attention and upkeep whilst also being difficult to automate.
In an ideal world, investigations in virology would be performed using a ‘black box’ of molecular diagnostic assays, but the reality is usually more complicated, comprising either in-house testing methods or commercially available assays. The decision about whether to use a commercial or in-house assay is usually driven by commercial test availability, cost and performance. Commercial tests can be expensive and may require the acquisition of additional specialised technology. However, it is often simply not viable for laboratories to introduce in-house assays in place of well-established and well-performing commercial methods such as Bayer’s VERSANT branched DNA (bDNA) viral load measurement technology. In this context, delegates at the meeting had the opportunity to preview Bayer’s new automated VERSANT 440 Molecular System (pictured) which offers complete bDNA automation, significantly improving laboratory workflow. Enhancements to the bDNA assay menu for this new system will include introduction of a simultaneous HIV and HCV assay and enhanced sensitivity for HCV.
Development of assays in-house has the advantage that laboratories can control all aspects of design, thus maximising equipment usage. Such assays are also cheaper than commercial methods, and can be modified quickly if required - but they take longer to develop and introduce into service. The need for extensive validation may also be regarded as a disadvantage, along with time-consuming quality control and assurance of components.
“Molecular diagnostics is a technology-driven laboratory science”, Dr Pantelidis told delegates at the Bayer meeting. “In order to understand current developments and see where future developments are going, we need to understand the processes inside the so-called ‘black box’ - i.e. extraction, amplification and detection.”
Extraction methods, he explained, have largely moved away from chemical methods to solid phase protocols, and some assays - such as those that utilise non-extracted lysis - avoid extraction altogether.

Inside the black box
There is now a wide diversity of methods available for amplifying targets, all of which have their own advantages and disadvantages. Basic in-house assays generally utilise PCR technology. This is because PCR is relatively cheap compared to other nucleic amplification methods, and is also easy to design. For increased sensitivity and specificity it may be linked to an end-stage hybridisation/ELISA or Real Time detection step. A single round PCR amplification is normally involved in routine diagnostics. Nested PCR assays - although more sensitive - are more prone to false positive results.
Similar diversity exists in terms of detection methods, and basic in-house assays typically involve a variety of these in the categories of electrophoresis, chromatography, hybridisation, enzymatic and mass spectrometry.
However, a unified approach to amplification and detection is now emerging. The majority of new commercial and in-house diagnostic tests combine amplification with detection in the form of real-time PCR technology. This approach provides users with the benefit of increased sensitivity and specificity for the detection of several different pathogens or genetic variants through use of a single technology platform. It is less suitable for multiplexing, however, and line-probe (LiPA) assays - such as Bayer’s VERSANT genotype tests - are considered better for multiple target screening. Such assays utilise a single amplification step for multiple targets, making them ideal for use when sample material is limited.
Real-time PCR methods detect amplified PCR products by the hybridisation of fluorescently labelled probes during amplification. Dr Pantelidis described the main advantage of such methods in terms of increased speed, thereby removing the need for post-PCR detection which would typically take between 30 minutes and one hour. “Real-time PCR can be used for both qualitative and quantitative analysis, as well as for genotyping”, he said, “and amplification time tends to be shorter. There is also the advantage of higher sensitivity compared with traditional PCR.”
Delegates learned that the current generation of automated extractors all use

User group meetings like this one at the Royal Opera House, London, provide the perfect opportunity to mix and network with peers in the scientific community

some form of chemical lysis, which limits the initial volume that can be extracted. However, ‘next generation’ extractors are moving away from small volumes measured in μL to relatively large starting volumes of 5-10mL, using alternatives to liquid lysis. There is also a trend away from the movement of liquid to moving nucleic acid captured in beads.
Looking into the future, Dr Pantelidis foresaw further advances in the areas of automated extraction, amplification and detection. In a separate presentation, delegates at the meeting also learned about Bayer’s plans to develop a flexible Kinetic PCR (kPCR) system. This will utilise proprietary homogeneous magnetic bead extraction technology on an automated platform, combined with a thermal cycler and software to achieve highly sensitive detection and quantitation through real-time PCR.

Developments in miniaturisation
Miniaturisation of technology is another interesting trend, with the prospect of an entire viral genome being contained within a single resequencing chip which would permit the identification of all emerging viral mutations. “This technology is particularly applicable to mutational screening, especially in respect of the variants which emerge in response to treatment”, said Dr Pantelidis.
Two major developments have taken place in the field of multiplexing and mass spectrometry, representing two alternative schools of thought. The first of these favours amplification of everything present in a sample, after which mass spectrometry can then be used to identify specific targets. This approach utilises Triangulation Identification for the Genetic Evaluation of Risks (TIGER) technology. The alternative approach favours the intelligent selection of multiple targets which in conjunction with Mass Tag PCR technology is used to achieve differential detection of pathogens.
Proponents of TIGER technology stress the rapidity of this method, as evidenced by its ability to identify an organism type within hours and a specific strain within 24 hours. The technique allows virtually all known, newly emergent and bioengineered pathogens to be identified in a single test, and is claimed to be less expensive than running thousands of individual tests for detection purposes. It is also a flexible approach, characterised by high throughput, multiple

Bayer’s new automated VERSANT 440 Molecular System which offers complete bDNA automation, significantly improving laboratory workflow

detection and identification of diverse samples.
At the Bayer meeting, Dr Pantelidis declared himself to be more in favour of the alternative Mass Tag PCR technology in a diagnostic setting due to its capacity for multiplex analysis. “Because organisms can be targeted with this method, a limited number of primers is required”, he explained. “This can help multiplex reactions since the limit of multiplexing is defined by the maximal primer concentration that can be accommodated in a PCR mix. Thus use of degenerate primers such as enteroviruses and adenoviruses can reduce sensitivity, but also increase coverage. Analysis requires the purification of PCR product from unincorporated primers and mass spectroscopy, but both of these processes can be automated.”

Exciting times ahead
Concluding his presentation, Dr Pantelidis expressed the view that the concept of black box diagnostics may well become a reality in the next few years, and the more distant possibility of having whole laboratories incorporated on a single chip means that there are exciting times ahead.
 After listening to a subsequent presentation by Bayer’s Sales Director for the UK and Ireland, Adrian Ralph, delegates were left in no doubt of Bayer’s commitment to the Molecular Diagnostics market. Acknowledging the requirement for laboratories to consolidate and simplify workflow whilst improving efficiency and maintaining results quality, he explained how Bayer is continuing to invest in developing its proprietary VERSANT bDNA automation and TRUGENE genotyping technologies as well as LiPA automation. The company also intends to invest in the development of genomic biomarkers to help predict the efficacy of specific therapies, working in partnership with academia and other companies in the pharmaceutical sector. The future looks exciting indeed.

By Dr Pantelidis